Multiplex Gene Editing and Effect Analysis of Yield, Fragrance, and Blast Resistance Genes in Rice

Background: The coordinated improvement of yield, quality and resistance is a primary goal in rice breeding. Gene editing technology is a novel method for precise multiplex gene improvement. Methods: In this study, we constructed a multiplex CRISPR/Cas9 vector targeting yield-related genes (GS3, OsPIL15, Gn1a), fragrance gene (OsBADH2), and rice blast resistance gene (Pi21) to pyramid traits for enhanced yield, quality, and disease resistance in rice. A tRNA-assisted CRISPR/Cas9 multiplex gene editing vector, M601-OsPIL15/GS3/Gn1a/OsBADH2/Pi21-gRNA, was constructed. Genetic transformation was performed via Agrobacterium-mediated method. Mutation editing efficiency was detected in T0 transgenic plants. Grain length, grain number per panicle, 2-acetyl-1-pyrroline (2-AP) content, and rice blast resistance of homozygous lines were measured in the T2 generations. Results: Effectively edited plants were obtained in the T0 generation. The simultaneous editing efficiency for all five genes reached 9.38%. The individual gene editing efficiencies for Pi21, GS3, OsBADH2, Gn1a, and OsPIL15 were 78%, 63%, 56%, 54%, and 13%, respectively. Five five-gene homozygous edited lines with two genotypes were selected in the T2 generation. Compared with the wild-type (WT), the edited homozygous lines showed increased grain length (2.46%–8.62%), increased grain length-width ratio (3.31%–5.67%), increased grain number per panicle (14.47%–27.11%), a 42–64 folds increase in the fragrant substance 2-AP content, and significantly enhanced rice blast resistance. Meanwhile, there were no significant changes in other agronomic traits. Conclusions: CRISPR/Cas9-mediated multiplex gene editing technology enabled the simultaneous editing of genes related to rice yield, quality, and disease resistance. This provides an effective approach for obtaining new japonica rice germplasm with blast resistance, long grains, and fragrance.